Glioblastoma multiforme (GBM) represents the most common primary malignant tumor of the adult central nervous system. The median survival after surgical intervention alone is approximately six months and the addition of radio-/chemotherapy can extend this time up to twelve months. Failed therapy is most often associated with local recurrence in the proximity of the original tumor. Consequently, efforts aimed at developing new therapies have focused on treatment strategies that target the tumor environment but spare normal and healthy surrounding brain cells. Oncolytic viruses (CRAds) that are replication competent in tumor but not in normal cells represent a novel approach for treating neoplastic diseases. To bypass the dependence on CAR for adenoviral entry and replication, we created a new generation of novel adenoviral vectors which selectively bind to receptors which are over-expressed by GBMs. First, we substituted the receptor binding domain of Ad5 with either (a) integrin binding sequence, RGD, (b) the receptor binding domain of serotype Ad3, or (c) a heparan sulfate binding proteoglycan (HSPG), pk7. Second, we tested a variety of tumor specific promoters (TSP) and identified survivin as the optimal TSP for transcriptional control of E1a. Survivin expression in gliomas is associated with poor prognosis, increased rates of recurrence, and resistance to chemo- and radiotherapy. Finally, in an effort to create a novel viral imaging modality that would provide a signal correlating with viral replication and progeny production, we developed an approach to incorporate an imageable substrate directly into the adenovirus capsid for subsequent imaging of viral replication and spread with PET. Based on the above considerations, we then created three conditionally replicative vectors: CRAd-S-RGD, CRAd-S-5/3, and CRAd- S-pk7. Our hypothesis is that such transcriptional/transductional modifications of Ad5 will show superior tumor selectivity and a high oncolytic efficacy against malignant glioma, with minimal toxicity to normal brain. Preliminary studies suggest that of the three vectors, CRAd-S-pk7 is the optimal virus for further preclinical development. In this application, we will focus on CRAd-S-pk7 and examine the mechanism by which this novel virus mediates enhanced glioma oncolysis in vitro and in vivo by pursuing the following specific aims: (1) characterize the infectivity, replication, and gene expression profile of CRAd-S-pk7 in primary glioblastoma multiforme in vitro; (2) determine if CRAd-S-pk7 exhibits enhanced oncolysis efficacy in vivo; (3) compare the toxicity, blood clearance, biodistribution, and immune profile of CRAd-S-pk7 between a fully permissive and semi-permissive animal model in vivo; and (4) determine whether CRAd-S-pk7 labeled with HSV-TK can be used for assessing viral replication and response to oncolytic virotherapy with PET in vivo. Malignant brain tumors remain highly resistant to conventional therapy and new and novel treatments are urgently needed to make an impact on this devastating disease. The aim of this proposal is to complete the preclinical studies of a novel oncolytic adenovirus with the aim of rapidly translating the results into a phase I/II clinical trial.